ben
September 30, 2019, 1:21am
4
Did you BLAST any of these sequences?
I primarily evaluate lung/bronchoalvolear lavage samples. They appear very similar to your samples. The unclassified ended up being eukaryotic DNA.
What I did was a strict filter with a qiime quality control step with 99% homology to Greengenes and this filtered out all non classified and bacterial DNA only classified at the kingdom level.
Yes, from my experience they are from eukaryotic DNA which for some reason has homology with the 16S primers.
There is a quality control step, which requires a Vsearch filter to remove anything that doesn't hit with the 16S 97-99% homology with 16S which has dramatically cleaned up my samples.
edit: The clustering methods in QIIME1 has a "sort of" built in filtering step. Remember the usearch in QIIME1 requires 97% match either open or closed clustering for it to "hit". DADA2 does not do this,…
Please see what this post did:
Hi folks, I'm looking for some help in troubleshooting the source of error in a recent analysis I did that is turning up with large numbers of unassigned features and even more assignments only at the Kingdom level (as seen in the barplots here )
Summary: 16s, mouse colon, 2x300 paired-end, MiSeq run using V3-V4 primers (341F & 805R). q2 version 2017.12.
I denoised using DADA2 with somewhat lenient truncating parameters based on the QC plots which can be found in the provenances. It ran success…
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